Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonist-bound conformation

Assaf Mosquna, Francis C. Peterson, Sang Youl Park, Jorge Lozano-Juste, Brian F. Volkman, Sean R. Cutler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

82 Scopus citations

Abstract

Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gainof- function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta.

Original languageAmerican English
Pages (from-to)20838-20843
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number51
DOIs
StatePublished - 20 Dec 2011
Externally publishedYes

Keywords

  • Constitutively active receptor
  • Pyrabactin resistance 1-like
  • Regulatory component of abscisic acid receptor
  • Seed dormancy
  • stAR-related lipid transfer domain

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